Classically activated or inflammatory macrophages are characterized by enhanced microbial killing, production of inflammatory cytokines such as TNF, IL-1 and IL-6, and high level production of IL-6/IL-12 family cytokines (IL-6, IL-12, IL-23 and IL-27) that regulate acquired immune responses. Inflammatory macrophages and their cytokine products play a key role in the pathogenesis of many autoimmune/inflammatory diseases, including rheumatoid arthritis (RA), inflammatory bowel disease and atherosclerosis, and possibly systemic lupus erythematosus (SLE). This application will focus on mechanisms that selectively regulate expression of specific subsets of inflammatory macrophage genes. Selective regulation of subsets of macrophage genes allows fine-tuning of distinct effector functions. Our long term goals are to understand mechanisms that selectively regulate macrophage effector functions important in human autoimmune and inflammatory diseases and to utilize this knowledge in development of new therapeutic approaches. We are particularly interested in regulation of activation and function of human monocytes as they enter inflammatory sites and differentiate into inflammatory macrophages, as this activation process is highly relevant for inflammatory disease pathogenesis. Therefore, we have initiated studies to identify mechanisms that selectively regulate human monocyte/macrophage activation in response to the prototypic macrophage-activating receptors TLR4 and TLR2. These TLRs elicit strong macrophage activation that serves as a good model for studying macrophage responses to inflammatory factors, including cytokines such as IL-1 and TNF that activate similar pathways. In addition, TLRs have been directly implicated in many autoimmune diseases, likely secondary to mediating responses to damaged tissues or cells (for example TLR2/4 in arthritis and TLR3/7/8/9 in lupus). We have identified a specific subset of TLR-inducible genes that are synergistically activated by TLRs and the Notch signaling pathway. Notch pathway components and target genes are expressed during inflammation, including in arthritis and atherosclerosis. TLRs directly activated Notch- dependent genes by IKK- and p38-mediated pathways, including activation of the transcriptional repressors Hes1 and Hey1 that selectively regulated expression of IL-6, IL-12 and IL-27. In this application, we will investigate molecular mechanisms by which TLRs and Notch synergize to activate expression of macrophage effector genes and the mechanisms and (patho)physiological significance of cytokine gene regulation by Hes1 and Hey1. We will use human systems that are directly relevant for inflammatory disease pathogenesis and murine models to further address the in vivo significance of our findings. We anticipate that our studies will yield insights into selective regulation of macrophage effector functions that can be exploited for therapeutic interventions to suppress inflammation in autoimmune diseases.